Mitochondria-targeted Nanoparticles Multimodal Imaging And Sonodynamic Therapy For Breast Cancer

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PREPARATION OF MITOCHONDRIA-TARGETED NANOPARTICLES IR780-NDS AND DETECTION OF THEIR BASIC PROPERTIESObjective To prepare a lipid nanoparticle(IR780-NDs)coated with perfluoropentane(PFP)and containing IR780,and to investigate its morphology,particle size,potential,encapsulation rate and production capacity of reactive oxygen species(ROS).Methods IR780-NDs was prepared by rotation-emulsification method.The size and morphology of nanoparticles were observed by light microscope and transmission electron microscope.Particle size and potential were detected by Malvern particle size analyzer.By drawing the standard curve of IR780,the envelop rate of IR780 in IR780-NDs were calculated.Reactive oxygen generation capacity was evaluated by oxygen sensor green reagent(SOSG).Results The IR780-NDs aqueous solution were pale green,uniform in size and dispersion under light microscope,and uniform in circle under projection electron microscope.Malvern particle size analyzer detected that the average particle size was(354.5 ± 156.3)nm and the potential was-(17.95)m V.The encapsulation rate was 82.6%.Reactive oxygen species(ROS)can be produced by IR780-NDs irradiated by ultrasound.Conclusion The lipid nanoparticles IR780-NDs loaded with PFP and IR780 have been successfully prepared in this study.IR780-NDs were round in size and distributed uniformly,and can produce reactive oxygen species after ultrasonic irradiation.PART ? MITOCHONDRIAL-TARGETED NANOPARTICLES IR780-NDS MULTIMODAL IMAGING AND IN VIVO BIOLOGICAL DISTRIBUTIONObjective Research IR780-NDs in vivo and in vitro Fluorescent imaging,Photoacoustic imaging and ultrasound imaging to evaluate the feasibility of it as a multimodal contrast agents,observe IR780-NDs of biological distribution,evaluation of its priority in tumor tissue clustering;To evaluate the biosafety of IR780-NDs.Methods IR780-NDs solution of 2 mg/ml and PBS solution of 100 ?L each were placed in the 96-well plate,and the fluorescence imaging effect was observed with the in vivo fluorescence imager.IR780-NDs solutions with different concentrations were used to observe the effects of photoacoustic imaging in Vevo Laser photoacoustic imaging system.IR780-NDs solution of 2 mg/ml was prepared and subjected to ultrasound irradiation for 3 min at different intensities(0.8 W/cm2,1.6 W/cm2,2.4 W/cm2,3.0 W/cm2,4.0 W/cm2).Ultrasound diagnostic instrument was used to observe the effect of enhanced ultrasound imaging.After collecting fluorescence/photoacoustic/ultrasonic images,the signal strength of region of interest(ROI)of each image was analyzed to evaluate the effect of IR780-NDs multi-mode imaging in vitro.The xenograft tumor model of 4T1 breast cancer in nude mice was established.When the tumor volume increased to about 100 mm3,the multimodal imaging experiment of fluorescence/photoacoustic/ultrasound in vivo and the biological distribution in vivo were conducted.In the in vivo fluorescence imaging experiment,IR780-NDs(concentration 3 mg/ml)were injected through the tail vein,and fluorescence images of tumor sites at different time points(0 h,0.5 h,1 h,2 h,4 h,6 h,24 h)were collected after administration.After 24 hours,tumor bearing mice were sacrificed to disintegrate tumor tissues and important organs for fluorescence imaging.After the Di I labeled NDs and IR780-NDs were injected into the tail vein,the tumor tissues and important organs of the tumor-bearing mice were sacrificed 24 hours later for ultrathin sections,and the nuclear DAPI staining was performed.The distribution of the two groups of nanoparticles in vivo was observed under a fluorescence microscope.In vivo photoacoustic imaging,IR780-NDs(concentration 3 mg/ml)were injected via tail vein,and photoacoustic imaging of tumor site was performed at different time points(0 h,1 h,6 h,24 h)after administration.In in vivo ultrasound imaging,IR780-NDs(concentration :3 mg/ml)were injected via tail vein,and the tumor site was irradiated by ultrasound 24 hours after administration.Ultrasound imaging before and after irradiation were observed by ultrasound diagnostic instrument.After collecting fluorescence/photoacoustic/ultrasonic/image,analyze the signal strength of region of interest(ROI)of each image.Random take 25 BALB/c mice were divided into control group,3 d,7 d,14 d,30 d group(n = 5),3 d,7 d and 14 d,30 d group by tail intravenous IR780-NDs(200 ?L,3.0 mg/m L),3 d,7 d after the treatment,14 d,30 d acquisition groups determination of blood for blood routine and biochemical indexes in mice,mice and dissociation important organ pathological HE staining.Results In vitro fluorescence imaging,the fluorescence intensity of IR780-NDs group was significantly stronger than that of PBS group.In vitro photoacoustic imaging,the photoacoustic signal of IR780-NDs increased with the increase of its concentration.In vitro ultrasound imaging,IR780-NDs can enhance ultrasound imaging by ultrasound irradiation,and the imaging effect is the best when the irradiation intensity is 2.4 W /cm2 for 3min.In the 4T1 breast cancer transplanted tumor model,fluorescence imaging in vivo showed that the fluorescence signal intensity of tumor site gradually increased with the extension of time and reached the maximum value 24 hours after drug administration.In the fluorescence imaging of tumor tissue and organs to be removed from body weight,the fluorescence signal of tumor tissue was significantly stronger than that of other important organs.In the ultrathin sections of tumor tissues and important organs,a large number of nano-particles in IR780-NDs group were clustered in tumor tissues,while in the NDs group,a large number of nano-particles were clustered in liver and spleen.In vivo photoacoustic imaging also showed that the intensity of the photoacoustic signal at the tumor site gradually increased with the extension of time and reached the maximum 24 hours after drug administration.In vivo ultrasound imaging showed that the ultrasound development of the tumor site was significantly enhanced after ultrasound irradiation.Conclusion Blood routine,blood biochemistry and pathological sections of vital organs(heart,liver,spleen,lung and kidney)of IR780-NDs mice in each group showed no significant difference with those in the control group in HE staining.PART ? MITOCHONDRIAL-TARGETED NANOPARTICLES IR780-NDS TARGETED AND SONODYNAMIC THERAPYObjective To evaluate the characteristics of 4T1 cells where IR780-NDs preferentially clustered in breast cancer,mitochondrial targeting ability,and penetrability of the tumor cell globule in 3D tumor cell spheres.To evaluate the intracellular ROS production capacity of IR780-NDs.To evaluate the efficacy and safety of sonodynamic therapy with ir78-nds on breast cancer cells and tumors.Method Breast cancer 4T1 cells were co-incubated with Di I labeled NDs and IR780-NDs(1 h,2 h,3 h,4 h),and the uptake of nanoparticles by breast cancer 4T1 cells was evaluated by confocal laser microscopy and flow cytometry.The 4T1 cells were co-incubated with Di I labeled NDs and IR780-NDs for 4h,Mito Tracker was used as the mitochondrial marker to observe the targeting ability of mitochondria of nanoparticles through a laser confocal microscope.3D spheroid of tumor cells were cultured and co-incubated with Di I labeled NDs and IR780-NDs for 12 h.The penetration of nanoparticles in 3D tumor cell spheres was observed by confocal laser microscopy.4T1 cells were divided into 5 groups: group I: control group,group II: US-only group,Group III: IR780-NDs-only group,Group IV: IR780-NDs+US group,and group V: positive control group.After different treatments,DCFH-DA was used as ROS probe to evaluate the intracellular ROS production capacity.After 4T1 cells were incubated with IR780-NDs at different concentrations(Concentration of the IR780 0 ?g/m L,1 ?g/m L,2 ?g/m L,3 ?g/m L,4 ?g/m L,5 ?g/m L)for 4 h,the viability of 4T1 cells was determined by CCCK-8.4T1 cells were incubated with IR780-NDs of different concentrations(Concentration of the IR780 0 ?g/m L,1 ?g/m L,2 ?g/m L,3 ?g/m L,4 ?g/m L,5 ?g/m L)for 4 h,and were irradiated with ultrasound(2.4 W/cm2,90 s)to detect the apoptosis of 4T1 cells by flow cytometry.The 4T1 cells were divided into four groups: group I: control group,group II: US-only group,Group III: IR780-NDs-only group,Group IV: IR780-NDs+US group.The 4T1 cells were stained with calcium xanthocyanin and pyridine iodide to observe the dead cells.A 4T1 breast cancer transplantation model was established,and treatment was started when the tumor volume increased to 100cm3.Tumor-bearing nude mice were randomly divided into four groups(n=5),group I: control group,group II: US-only group,Group III: IR780-NDs-only group,Group IV: IR780-NDs+US group.The tumor size was measured and the body weight of nude mice was weighed.After treatment,a nude mouse was sacrificed in each group to dissociate tumor tissue and important organs(heart,liver,spleen,lung and kidney).The tumor tissue were weighed and photographed,and pathological sections were stained with HE,TUNEL and PCAN immunohistochemical staining.Pathological sections of important organs were stained with HE.Results Laser confocal microscopy showed that 4T1 cells co-incubated with NDs and IR780-NDs at different time points(1 h,2 h,3 h,4 h),and the red fluorescence(representing nanoparticles)around 4T1 cells in IR780-NDs group was significantly stronger than that in NDs group,and the red fluorescence was stronger with the extension of time.Fluorescence quantitative analysis and flow cytometry also showed that the fluorescence intensity of IR780-NDs group was significantly higher than that of NDs group,and the difference was statistically significant(P<0.05).Confocal laser microscopy showed a large amount of red fluorescence(IR780-NDs)in the mitochondrial area of the IR780-NDs group,and the Pearson correlation(PC)=0.87.No obvious red fluorescence(NDs)was observed in the mitochondrial area of NDs group,and the Pearson correlation(PC)=0.15. confocal laser scanning microscopy showed that the nanoparticles in the NDs group only attached to the surface of the 3D spheroid,and the nanoparticles in the IR780-NDs group penetrated to the core of the 3D spheroid and distributed throughout the sphere.Confocal laser microscopy showed that the green fluorescence intensity of the positive control group was the highest in the intracellular active oxygen test.In addition,only the IR780-NDs+US group showed obvious green fluorescence,while no obvious green fluorescence was observed in the other groups.The viability of 4T1 cells decreased with the increase of IR780-NDs concentration and the extension of ultrasonic irradiation time.The apoptosis rate of 4T1 cells increased with the increase of IR780-NDs concentration.In the staining of living dead cells,the control group all had no cell death,while the US-only group and IR780-NDs-ony group only had a small amount of cell death,and the IR780-NDs+US group had a large amount of cell death.In vivo treatment results showed that tumor growth was not inhibited in the US-only group and IR780-NDs+US group,and there was no significant difference between the two groups(P<0.05).There was no significant difference in weight fluctuation between the treatment group and the control group.There was no significant difference in cell morphology between the HE staining US-only group of tumor tissue and the IR780-NDs-only group and the control group.Most cells in the IR780-NDs+US group had no complete structure,and the nuclear morphology changed(nuclear pyknosis,rupture and dissolution).Immunohistochemical staining results of TUNEL and PCNA showed that there were a large number of proliferation cells in the control group,only a small number of apoptotic cells in the US-only group and the IR780-NDs-only group,and large number of apoptotic cells in the IR780-NDs + US group.Conclusion IR780-NDs can be preferentially clustered around breast cancer 4T1 cells with good mitochondrial targeting,and can penetrate into the ball core of 3D spheroid of tumor cells.IR780-NDs can kill tumor cells and inhibit tumor growth through sonodynamics with high therapeutic safety.